"Premalignant" breast lesions are both risk factors and precursors of invasive breast cancer [IBC]. The evolutionary stages of these lesions include typical hyperplasia [TH], atypical hyperplasia [AH], and carcinoma in situ [CIS]. Clinically, it would be very important to identify biomarkers able to distinguish lesions likely to progress from those which will not, in order to take appropriate preventive measures. Furthermore, a knowledge of the molecular correlates of these stages might provide targets for focusing these preventive measures. We have therefore assembled a large evolutionary tumor bank of archival breast tissues representing these stages. Using immunohistochemistry [IHC], we have shown that lesions of all stages proliferate faster than normal breast epithelium, independent of hormonal control, and also have altered rates of apoptosis. We are able to distinguish three grades of CIS based on five IHC markers (ER and PgR, p53, c-erbB-2, and Ki67) as well as on histomorphology. We have also developed microdissection techniques suitable for isolating DNA from these archival specimens for allelic imbalance (loss-of- heterozygosity) studies, and RNA for differential display studies, from relatively pure lesions. These techniques have already allowed us to sow that there is a clonal progression from one stage to the next in lesions of different stages in the same breast. Finally, as spin-offs of this project, we have found evidence of a gene required for metastasis near a tumor suppressor gene on chromosome 14q, forming the basis for the new Project 4, and have discovered in several TH lesions the same super-active estrogen receptor polymorphism, which may therefore be important in the development of premalignant breast disease. To pursue the goals of identifying markers of risk and evolutionary progression in premalignant breast lesions, we now propose: (1) To identify growth regulatory factors, including receptors along with proliferation and apoptosis markers, which are associated with the development and progression of human premalignant breast disease and which are clinically associated with risk of developing invasive breast cancer [IBC]; (2) to identify invasion-related factors, including certain adhesion molecules and the urokinase-type plasminogen activator system, which are associated specifically with progression of CIS to IBC; and (3) To identify, by differential display and appropriate RT-PCR and immunohistochemical confirmation, new gene products whose loss or gain is associated with progression of premalignant lesions.